Address to: Dr.Luís Marcelo Aranha Camargo. ICB5/USP. Rua Francisco Prestes, 2827, Setor II, 76888-000 Monte Negro, RO, Brasil.
Phone: 55 69 3530-2349 e-mail: [email protected] Received 15 July 2012 Accepted 14 February 2013
http://dx.doi.org/10.1590/0037-8682-1708-2013
Major Article
INTRODUCTION
Survey of Bancroftian ilariasis infection in humans and
Culex
mosquitoes in the western Brazilian Amazon region:
implications for transmission and control
Rodolfo Luís Korte
[1],[2], Gilberto Fontes
[3],
Juliana de Souza Almeida Aranha Camargo
[1],
Eliana Maria Maurício da Rocha
[3], Edicarlos André Cavalcante de Araújo
[1],
Marcelo Zagonel
de Oliveira
[4], Rafael Vital dos Santos
[5]and Luís Marcelo Aranha Camargo
[1],[6][1]. Departamento de Medicina, Faculdade São Lucas, Porto Velho, RO. [2]. Departamento de Medicina, Universidade Federal de Rondônia, Porto Velho, RO. [3]. Campus Centro Oeste, Universidade Federal de São João del Rei, Divinópolis, MG. [4]. Departamento de Genética, Universidade Federal do Rio Grande do Sul, Instituto Nacional de Genética Médica Populacional, Porto Alegre, RS. [5]. Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Alagoas, Maceió, AL. [6]. Instituto de Ciências Biomédicas 5, Universidade de São Paulo, Monte Negro, RO.
ABSTRACT
Introduction: The aim of this work was to identify possible lymphatic ilariasis foci in the western Brazilian Amazonian that could be established from the reports of Rachou in the 1950s. The study was conducted in three cities of the western Brazilian Amazon region - Porto Velho and Guajará-Mirim (State of Rondônia) and Humaitá (State of Amazonas). Methods: For human infection evaluation thick blood smear stained with Giemsa was used to analyze samples collected from 10pm to 1am. Polymerase chain reaction (PCR) was used to examine mosquito vectors for the presence of Wuchereria bancrofti DNA. Humans were randomly sampled from night schools students and from inhabitants in neighborhoods lacking sanitation. Mosquitoes were collected from residences only. Results: A total 2,709 night students enrolled in the Program for Education of Young Adults (EJA), and 935 people registered in the residences near the schools were examined, being 641 from Porto Velho, 214 from Guajará-Mirim and 80 from Humaitá. No individual examined was positive for the presence of microilariae in the blood stream. A total of 7,860 female
Culex quinquefasciatus specimens examined were negative by PCR. Conclusions: This survey including human and mosquito examinations indicates that the western Amazon region of Brazil is not a focus of Bancroftian ilariasis infection or transmission. Therefore, there is no need to be included in the Brazilian lymphatic ilariasis control program.
Keywords: Lymphatic ilariasis. Bancroftian ilariasis. Amazon Region. Wuchereria bancrofti. Culex quinquefasciatus.
Lymphatic ilariasis, caused by Wuchereria bancrofti, is considered a neglected disease that affects the lives of people in many endemic regions of Ásia, Latin America, and África. The parasite is transmitted by Culex quinquefasciatus, especially in socially and economically depressed communities with no sanitation systems or treated water.
Currently, more than 1.3 billion people are at risk of the disease in 72 endemic countries. Approximately 65% of those people live in southeastern Ásia and 30% in Africa, with the remainder in other tropical areas of the world1-3. In the
Americas there are active foci in Brazil, Guyana, Haiti and the
Dominican Republic, with 12.4 million people requiring mass drug administration. Haiti accounts for the highest proportion2.
The only focus of lymphatic ilariasis known in Brazil is the metropolitan area of Recife (State of Pernambuco)3. Cities such
as Belém (State of Pará) and Manaus (State of Amazonas), in the Amazon region, and Maceió (State of Alagoas), in Northeast Brazil, have been described as endemic areas in the past. However, these foci have been extinguished and are presently kept under surveillance3,4.
In 1953, a hemoscopic survey was performed in Porto Velho, State of Rondônia, Brazil, in which 1,400 individuals were examined (12% of the population) and 6 (0.4%) were positive for W. bancrofti microilariae5. However, none of these
individuals was autochthonous but were, rather, migrants from areas known to be endemic at the time, such as Belém (PA) and Manaus (AM), who had been living in Porto Velho for a shorter time than the parasite's prepatent period5.
In 1953, in State of Rondonia, another hemoscopic survey was performed in Guajará-Mirim. A total of 603 individuals were examined, and only one (0.2%) was positive for W. bancrofti
METHODS
performed in Humaitá (AM), but none of the 435 individuals examined was found infected6.
Porto Velho has never been an endemic area of lymphatic ilariasis, unlike other Amazonian cities such as Manaus and Belém, which were foci of active ilariasis transmission.
The existence of many unconirmed reports of elephantiasis cases in Porto Velho, the lack of further epidemiological surveys after those performed by Rachou et al.5 in the 1950s, and the high
density of C. quinquefasciatus prompted the present research, which was conducted to examine the actual occurrence of this endemic disease in that region.
Survey of human infection/inclusion and exclusion criteria
Individuals under the age of ive years old were excluded, as the hypothesis considered that a short time of exposure to infection would not justify the examination. Individuals who refused to participate in the survey were also excluded. All remaining individuals aged ive years and over participated in the survey.
The criteria used to select the places to be evaluated included areas populated earlier in the studied cities, exhibiting urban poor characteristics with respect to their infrastructure, basic sanitation, open-air sewage and houses built on wood pilings, which met the conditions required for vector growth.
Study areas
Domicile survey in the Municipality of Porto Velho: The peripheral area and older neighborhoods of Porto Velho (8° 45’ 42.57” S, 63° 54’ 07.06” W) were the target areas of the study alongside the Madeira River, where there are slums and houses built on pilings. The neighborhood and residences of Porto Velho were randomly sampled. The sample size was calculated after mapping and a census of the neighborhoods by taking each dwelling as a sample unit, using an empirical prevalence of 1%, a design effect for cluster surveys of 1.8 and a conidence interval of 95% calculated by OpenEpi7. In this case, the sample size was estimated as a total of 557 randomly selected dwellings and 974 individuals older than ive years old. To perform the study, the selected neighborhoods were divided into seven sectors, and 1/3 of the houses in each section were randomized and selected. Domicile survey in the Municipality of Guajará-Mirim: All dwellings in the old neighborhood of Triângulo in Guajará-Mirim (10° 47’ 27.33” S, 65° 19’ 55.98” W) were selected because they shared the same poor urban infrastructure characteristics as the areas of Porto Velho. The population of Triângulo was estimated to comprise 506 individuals of all ages, and a total of 214 (31.7%) blood samples were collected. Individuals more than ive years old living in the selected area participated in the survey. The sample was not randomized and was intended to comprise all the inhabitants and houses.
Domicile survey in the Municipality of Humaitá, State of Amazonas: All dwellings of the old neighborhood of Santo Antonio in Humaitá (7° 30’ 22.20” S, 63° 01’ 37.82” W) were
selected, with 80 houses featuring a similar infrastructure as those in the previously described areas and with a population of 240 inhabitants. The sample was not randomized and was intended to comprise all houses and inhabitants.
Figure 1 shows the areas of research for W. bancrofti in the western Amazonian region and the old and present foci of lymphatic ilariasis in Brazil (Figure 1).
Survey of night students
Since evening students include mainly adolescents and young adults, the highest-risk group for bancroftosis8, and
because of the parasite's nocturnal periodicity in the region9,
the survey was also performed in public night schools in Porto Velho, Guajará-Mirim, and Humaitá. All 23 schools, which have a total of 3,601 students in the target area, were located within the oldest risk areas of the three cities involved in the Program for Education of Young Adults (EJA). These schools provide education to any student, youth and adult older than 18. All the students who wanted to participate were included in the sample.
Blood samples and diagnosis
The blood samples were collected from 10pm to 1am in the dwellings and from 10 to 11pm in the schools due to the nightly periodicity of the microilariae in the peripheral blood stream of the human hosts9. The thick blood smear (TBS) method was
used for the diagnosis according to the following protocol: blood collection was performed by inger prick using a disposable lancet and used for the preparation of TBS equivalent to 0.06-0.08mL of blood, which were dried at room temperature. Twelve hours after being dried, the TBSs were dehemoglobinized, ixed with methanol, stained with eosin at 0.05%, and then stained with Giemsa. The slides were examined under an optical microscope with an objective lens with magniications ranging from 10 to 40X in a blinded manner by two independent examiners.
Survey of mosquito infection
Vector collection: The Culicidae captures were performed in Porto Velho, Guajará-Mirim and Humaitá in the neighborhoods of the humans who participated in this study. All the dwellings were visited and registered except locked dwellings and those whose owners refused to participate in the survey. The number of visited dwellings exceeded the originally sampled dwellings to increase the number of mosquitoes captured. An aspirator was used to collect adult mosquitoes (Castro aspirator). Ingurgitated insects were captured inside the houses from 7 to 10am. The insects were placed inside special ine mesh cages, separated according to the collection location and date, and then taken to the laboratory. The captured C. quinquefasciatus females were selected, placed inside test tubes (ive mosquitoes/tube), and frozen at -20oC for further polymerase chain reaction(PCR).
Detection of W. bancrofti in mosquitoes by polymerase chain reaction (PCR): Briely, genomic DNA was isolated from blood-engorged mosquitoes using the method described by Vasuki et al.10 with minor modiications. Dried mosquitoes
RESUlTS
FIGURE 1 - Map showing the survey locations (Porto Velho, Guajará-Mirim and Humaitá), the old foci of lymphatic ilariasis transmission where the disease has already been eliminated (Belém, Manaus and Maceió), and the only area where lymphatic ilariasis transmission is still present (Recife and its metropolitan area).
using a Microcon YM-100 centrifugal ilter device (millipore) according to the manufacturer's instructions, a step introduced in the original technique in order to concentrate the extracted DNA. The PCR was conducted using the primer pair NV1 (5’- CGT GAT GGC ATC AAA GTA GCG - ‘3) and NV2 (5’- CCC TCA CTT ACC ATA AGA CAA - ‘3) to detect the 188bp target repeat fragment (SspI) of W. bancrofti (SspI; GenBank accession n. L20344)11. Conventional PCR were performed in a
volume of 25µL containing 20pmol of each primer (NV1/NV2), 200 µM deoxyribonucleotide triphosphates (dNTPs), PCR buffer (50 mM KCl, 10mM Tris-HCl, pH 8.4), 1.5mM MgCl2, ultrapure DNA-free water, 1.5U/µL Taq DNA polymerase and 4µL of template DNA. All reagents were from Invitrogen®. The PCR thermal cycling (PCR Express ThermoHybaidTM)
conditions were 35 cycles of 92°C for 1min, 55°C for 1min, and 72°C for 1min. The PCR amplicons were run on a 1.5% (m/v) agarose gel at 90V for 1h, stained with ethidium bromide (4mg/mL), and visualized under ultraviolet illumination (Vilber LourmatTM TFX-20.M). The gels were photographed using
a video documentation system (Vilber LourmatTM DP-001 FDC)
and then analyzed (photoCaptMW for Windows 10.01 Vilber LourmatTM).
Ethical considerations
Every person examined signed a consent form and was well informed before being engaged in the survey, and the participation of the minors was authorized by their parents or guardians. Every person examined was able to sign his/ her name. The project was approved by the Internal Review Board (IRB) of São Lucas College on December 11, 2007, and registered under the number 143/2007 and by the REC of São Paulo University (USP).
Human Infection
Domicile surveys in the Cities of Porto Velho, Guajará-Mirim and Humaitá: In Porto Velho, 493 houses were visited (88.5% of the total houses selected for inclusion), and 641
BRAZIL
72'0'0'W 63'0'0'W 54'0'0'W 43'0'0'W 36'0'0'W
0'0'0'
N
0'0'0'
N
2'0'0'
S
10'0'0'S
18'0'0'
S
26'0'0'
S
34'0'0'
S
72'0'0'W 63'0'0'W 54'0'0'W 43'0'0'W 36'0'0'W
18'0'0'
S
26'0'0'
S
34'0'0'
S
N
people were surveyed (65.8% of the individuals in the deined area). In Guajará-Mirim, 169 houses were visited (100% of the houses in the deined area), and 214 dwellers were examined (42.3% of the total individuals). In Humaitá, all 80 houses of the old neighborhood of the Santo Antonio Community were visited, and 80 individuals were surveyed (33.3% of the total individuals). In the three surveyed areas, all examined samples were negative for microilariae (Table 1).
Night students survey: Out of 3,601 students, 2,709 were examined in the three research areas. In Porto Velho, the visits involved 11 schools in the previously deined area and included 1,684 (71.1%) students. In Guajará-Mirim, all ive schools in the previously deined area were visited, and 272 (81.2%)
students were examined. In Humaitá, all seven night schools in the previously deined area were visited, and 753 (83.6%) students were examined (Table 2). All results were negative for microilariae in the students blood. This study found no evidence of microilarial infection in any of the individuals studied in all three areas (Table 2).
Survey of the infection of mosquito vectors: A total of 8,212 female mosquitoes were collected in the three target areas, with 95.8% (7,860) being C. quinquefasciatus and 4.2% belonging to other genera, including Aedes, Psorophora and Anopheles. PCR was performed to detect the DNA of W. bancrofti in all
C. quinquefasciatus females, but none of the mosquitoes was positive (Table 3).
TABLE 1 - Results of thick blood smear examination for Wuchereria bancrofti infection carried out among the population of three cities in the Amazon region of Brazil - 2008/2009
Number of houses in Visited houses Number of dwellers Examined dwellers** Result Municipalities the surveyed area n % in the surveyed area* n % positive exams
Porto Velho 557 493 88.5 974 641 65.8 0
Guajará-Mirim 169 169 100.0 506 214 42.3 0
Humaitá 80 80 100.0 240 80 33.3 0
Total 806 742 92.0 1,720 935 54.4 0
*All ages, including children under 5 year old; **People who accepted to participate in the survey over 5 years old.
TABLE 2 - Results of thick blood smear examinations for Wuchereria bancrofti infection carried out among students older than 18 years in schools, in three cities in the Amazon region of Brazil - 2009
Number of schools Total number Students examined Result
Municipalities enrolled in the study of students n % positive exams
Porto Velho 11 2,368 1,684 71.1 0
Guajará-Mirim 5 333 272 81.7 0
Humaitá 7 900 753 83.7 0
Total 23 3,601 2,709 75.2 0
TABLE 3 - Results of the screening ofCulex quinquefasciatusmosquitoes forWuchereria bancroftiDNA examined by polymerase chain reaction (PCR), collected in three cities of Brazilian Amazon region - 2008/2009
Number of Visited houses Houses - mosquitoes collection Total of female Culex Result
Municipality dwellings* n % n % mosquitoes n % positive PCR
Porto Velho 1,525 1,262 82.7 1,080 85.6 3,650 3,512 96.1 0
Guajará-Mirim 275 261 95.0 228 87.3 1,979 1,765 87.9 0
Humaitá 324 164 50.1 156 84.3 2,583 2,583 99.6 0
Total 2,124 1,687 79.4 1,464 86.8 8,212 7,860 95.8 0
PCR: polymerase chain reaction; *Some dwellings are not in the same area of the human survey, but in the neighbohood. In this case, the study area was ampliied in order to increase mosquito capture.
DISCUSSION
Since the 1960s, no survey has been performed to determine whether the vicinities of Porto Velho, Guajará-Mirim and Humaitá were or could be silent areas of lymphatic ilariasis transmission. The weather conditions of these areas in Amazonas
Using the TBS method, this work studied populations settled alongside the rivers in areas favorable for the development of this parasite as well as night students, whose environment conditions were similar to those reported in studies performed in extinct foci, such as Maceió and Belém3, and Recife
metropolitan region, the only remaining area of ilariasis in Brazil. No occurrence or evidence of the disease was found in the studied Cities of Rondônia and Amazonas.
In the present study, the diagnostic method was the TBS, and its main limitation is a reduced sensitivity to the identiication of patients affected by mild microilaremia12. However, TBS is
a rapid, practical and economic technique, commonly applied in large-scale ield studies, and features good sensitivity when the parasitemia is greater than 10 microilariae/mL of blood12.
This study, as similar surveys performed in other states, endeavored to provide care to the populations that are undoubtedly subjected to higher social and environmental risk factors, that is, extremely poor areas with no basic sanitation systems and that are close to areas of higher vector proliferation. Notably, the investigated municipalities are relatively new, whereas the surveys were performed in contiguous, older areas of these cities. Poor basic sanitation conditions in habitational conglomerates are determining factors that could foster and maintain ilariasis transmission. Thus, the populations exposed to higher social and environmental transmission risks were incorporated, and the likelihood of false-negative results for ilariasis infection was reduced. In addition to the risk population, the present study included also participants of the Young Adult Students’ program of the local State government involving 18 to 35 year old individuals enrolled in basic and mid-level schools. This categorization was chosen for operational reasons according to the studies performed previously in other areas of the country8,13. As suggested by Rocha et al.14, the prevalence
of bancroftosis may occur in young age brackets, which were emphasized in this survey. Furthermore, the surveyed schools were located near those places with a higher probability of the presence of the disease, which are the oldest settlements of the cities with poor urban infrastructure.
The assessment of ilariasis transmission was performed by monitoring the infection in the insect vectors by PCR. The PCR has proven to be a powerful tool to evaluate the contamination intensity of the mosquitoes in endemic areas and, because of these advantages, has replaced the conventional dissection methods for the diagnosis of W. bancrofti in mosquitoes15. This
method, in addition to being more sensitive than dissection, enables a large number of samples to be processed in a short period.
Other factors that may have contributed to the nonproliferation of the disease may be associated with the environment, the vector itself, and the cases diagnosed and treated in the 1960s.
Regarding the environment, ilariasis is known to occur in tropical and subtropical hot and humid areas. Chandra et al.16
found that the density of Culex mosquitoes decrease during rainy periods, although periods of higher temperatures and humidity favor W. bancrofti transmission, shortening developmental period from L1 to L3 (infective larva) stages of the parasite in the vector.
The studied areas of Porto Velho, Guajará-Mirim and Humaitá exhibit the characteristics that facilitate rapid development of the parasite into its infectious larval form. However, during periods of increased heat and humidity, the number of vectors decreases, although there is no synchrony between the infecting larvae and the number of vectors, and the transmission risk decreases. One of the factors that may have contributed to the nonproliferation of the disease is the lack of synchronization between the number of vectors and the environmental conditions for the development of the agent that causes the disease17.
Several vector types are involved in ilariasis transmission, including mosquitoes of the genera Culex, Anopheles, Aedes
and Mansonia18. In Brazil, the only known vectors are the
mosquitoes of the genus Culex, which are prevalent in the studied areas.
Studies with different ilariae worms show that the vector eficiency depends on the susceptibility to pathogen infection and the microilaria density19-21. Vectorial competence can be
affected by the densities of microilariae ingested during blood meal, and not all the blood fed mosquitoes became infected. Furthermore, when the insect is exposed to a high concentration of parasites an increasedvectorial mortality rate can occur.
The non occurrence of the disease may also be associated with the blood feeding in vectors of low vectorial competence. Not all larvae ingested by the mosquitoes will survive inside the gastrointestinal tract and some of the larvae are expelled by the vector itself, whereas others are injured by the oropharyngeal tract during the repast22. As a result, the
transmission is reduced22.
Those intrinsic and extrinsic factors and the complex interactions between ilarial parasites and mosquitoes, that inluence vectorial competence, could be in part responsible for geographic distribution of ilariasis.
Accordingly, the discussion also involves indings from the studies performed by Hairston and De Meillon23, which clearly
showed that immature W. bancrofti larvae do not survive inside their hosts, thereby largely decreasing the load of contaminative ilariae (L3) and increasing the number of bites required for infected mosquitoes to cause a parasitic infection. An estimated 15,500 infecting bites are required to cause microilaremia23.
Snow and Michael24 in 2002 and Chandra17 in 2008 also
showed that the ilariae present in the vector may increase the likeliness of dissemination but may also be fatal to the vector when their presence is too high by increasing the lethality, which contributes to the reduction in transmission.
the microilarial infection of the vertebrate hosts. The mosquito infection rate is proportional to the microilariae density in the peripheral blood stream of the patients. Brito et al.25 found
that the infection rate of mosquitoes fed with the blood of individuals with mild W. bancrofti microilarial infections (1-10 microilariae/mL) was 0.07%, whereas the eficiency of the vector was nearly 17%. That is, for every six microilariae ingested, only one reaches the infecting larva stage.
Several studies have demonstrated that the persistence of this endemic disease depends on the occurrence of high natural infection rates of the mosquitoes. Nevertheless, there has been no consensus among researchers that can forecast the occurrence of a new case of patent microilarial infection in humans26.
However, below a certain critical number of infecting bites, lymphatic ilariasis has not been conirmed to be an endemic disease, which may be the case in this area. In places where the microilariae density is maintained below ive microilariae/60mL blood, there is considered to be no transmission risk27.
In a recent study performed in Maceió it was showed that the ilarial load in a patient is one of the determining factors for maintaining transmission foci28. Such a load may have been one
of the factors that determined the disease dissemination in the studied areas. In Maceió, Leite et al.28 found that an individual
suffering from W. bancrofti microilarial infection who came from an endemic area and had been living for more than 10 years in a nonendemic area, was not capable of inducing the formation of a new bancroftosis focus, even though the new area exhibited the appropriate environmental conditions. One of the possibilities considered by the researchers was the mild microfilarial infection of the infected individual (4 microilariae/mL of blood)28.
However, with the growing migration in the country, there is a risk that the disease might be introduced in areas free of the infection3. In Sri Lanka infected migrants haveengendered
lymphatic ilariasis in areas where the disease was previously unknown29. This occurred also in metropolitan Recife, where
cases of the parasitosis appeared in previously unaffected areas30,31.
The magnitude of the potential risk for the establishment of sustainable foci of lymphatic ilariasis depends mainly on the number of W. bancrofti carriers, the microilarial density in these individuals, the existence of potential vectors, and environmental conditions that favor transmission32-34.
Finally, the few people suffering from W. bancrofti
microilarial infections found in the Cities of Porto Velho and Guarajá-Mirim in the 1950s had allochthonous, mild microilarial infections (out of the seven people infected with parasites, ive had fewer than ive microilariae per slide examined)5,35. It
appears that the small number of people infected with the parasites and the mild nature of the microilarial infection reported in the 1950s in Porto Velho and Guarajá-Mirim were not suficient to establish and maintain the lymphatic ilariasis in the examined areas, which were considered harmless and may or may not be associated with environmental and other factors related to the vectors.
ACKNOWlEDGMENTS
The authors declare that there is no conlict of interest. CONFlICT OF INTEREST
FINANCIAl SUPPORT
REFERENCES
The authors are especially grateful to the following for their support of this study: Dilene Morais B. da Silva, Célia Gonçalves Pereira, Beatriz Santos, and Suzane Neves Velasques. We acknowledge and give thanks to the students of São Lucas College, the population of the surveyed cities and the directors of the Program for Education of Young Adults (EJA).
Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) Proj no. 2007/00848-9.
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